Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (June et al., 2011). However, the particular combination of signaling domains, transmembrane and co-stimulatory domains used with respect to CD19 ScFv, was rather antigen-specific and cannot be expanded to any antigen markers.
The disialoganglioside GD3 is an acid glycosphingolipid depicted in FIG. 1 which has been shown to be strongly expressed in melanoma cell lines, adult and fetal brain and to a lesser extent in adult and fetal lung (Haraguchi et al, 1994; Nakayama et al. 1996). Gangliosides are a family composed of a common hydrophobic ceramide moiety and a hydrophilic oligosaccharide chain containing one or several sialic acids. Ceramide, therefore a source of GD3, is converted into GD3 by diverse glycosyltransferases and in particular by the GD3 synthase (alpha 2,8-sialyltransferase or ST8Sia I or SATII). GD3 ganglioside, also named alpha-N-acetylneuraminide alpha-2,8-sialyltransferase (ref: Uniprot: Q92185; SIA8A human), an enzyme that is encoded in humans by the ST8SIA1 gene (RefSeq: NP_003025.1. NM_003034.3).
GD3 is highly expressed at early development stages of the central nervous system, when neural cells proliferate actively. At later developmental stages, the GD3 content declines and others gangliosides become major species (Daniotti et al., 1997; Gravotta et al., 1989). In addition, the expression level of gangliosides in general, and GD3 in particular, is very low and restricted in adult extra neural tissues. Nevertheless, GD3 is highly expressed in tumor cells, accounting for more than 80% of melanomas. It is also overexpressed in neuroectodermal tumors (neuroblastoma and glioma) and carcinomas, including lung, breast, colon, prostate and ovary (Lo et al., 2010). In addition, GD3 expression was observed in T cell acute lymphoblastic leukemia (Reaman et al., 1990).
The highly restricted expression of GD3 on select tumor types and the fact that GD3 has been shown to promote tumorigenesis by mediating cell migration, adhesion, proliferation and differentiation (Birklé et al., 2003), makes it an attractive antigen for immunotherapeutic targeting. Previously, Junghans et al. engineered and validated anti-GD3scCARs containing the MB3.6 scFv (Lo et al., 2010; Yun et al., 2000). However, this CAR required systemic infusion of maximal dose of interleukin 2 (IL2) to reach 50% efficacy in the eradication of subcutaneous GD3 positive tumors in established nude mice tumor models. Thus, there is still a need of new anti-GD3 chimeric antigen receptors having improved efficacy, and which can be used for the treatment of both solid or liquid tumors.
The present inventors have thus considered that GD3 could be a valuable target antigen by using CAR-expressing T cells for treating liquid tumors, such as T cell acute lymphoblastic leukemia and solid tumors, such as melanomas, neuroectodermal tumors (neuroblastoma and glioma) and carcinomas, including lung, breast, colon, prostate and ovary.
As an alternative to the previous strategies, the present invention provides with new GD3 specific CAR constructs, which can be expressed in immune cells to target GD3 malignant cells with significant clinical advantage.